PROJECT SUMMARY/ABSTRACT This competing renewal application, U01 AA013521, Neuroimaging of Alcohol-Induced Neuroadaptation: Translation from Animals to Humans, is submitted in response to RFA-AA-16-004 (U01). The overarching aim of this proposal is to identify in vivo markers related to the pathogenesis of alcohol use disorder (AUD) related to altered stress (amygdala) circuitry and neuroimmune recruitment. Recent resting- state functional magnetic resonance imaging (rs-fMRI) studies will determine the differential circuitry of the amygdala as a function of withdrawal frequency in AUD. The potential for neuroimmune recruitment in AUD pathology will be evaluated using a multi-B-value diffusion tensor imaging (DTI) analysis of free water, which is proposed as an index of neuroinflammation. These in vivo biomarkers will be collected in humans, monkeys, and rodents and contribute to the validity of the animal models and provide a framework for evaluating pharmacological agents to aid in AUD recovery. Optogenetic stimulated functional MRI in rodents will further explore specific brain circuits relevant to AUD. Specific Aim 1 will use rs-fMRI and multi-B-value DTI in humans with AUD and their controls to test the hypothesis that greater withdrawal frequency will be associated with more symptoms of anxiety, changes in amygdala rs-fMRI connectivity (stronger functional connectivity between the CMA and anterior cingulate cortex [ACC] and between BLA and anterior insula; weaker connectivity between the CMA and posterior cingulate cortex [PCC]), and more free water in amygdala, hippocampus, and cingulate cortex. Specific Aim 2 will analyze rs-fMRI and multi-B-value DTI data in monkeys at baseline and following 3 cycles of EtOH exposure and withdrawal collected by INIA-Stress to test the hypothesis that greater withdrawal frequency will be associated with exacerbated anxiety, higher EtOH consumption, changes in amygdala rs-fMRI connectivity, and more free water in amygdala, hippocampus, and cingulate cortex. Specific Aim 3 will rs-fMRI and multi-B-value DTI in rats exposed to chronic intermittent ethanol (CIE) via vapor chambers to test the hypothesis that longer periods of CIE will result in the accrual of anxiety-like behavior, stronger functional connectivity between the amygdala and downstream targets (e.g., brainstem), and more free water in amygdala, hippocampus, and cingulate cortex. It is also hypothesized that apremilast, a phosphodiesterase-4 inhibitor will normalize affected behavioral, functional, and free water changes, thereby supporting neuroimmune mechanisms in AUD. Specific Aim 4 will use optogenetic and chemogenetic methods and fMRI to determine the neurocircuitry recruited by direct stimulation of the rat central nucleus of the amygdala (CeA) corticotropin releasing factor (CRF) neurons. The hypothesis to be tested is that direct stimulation of CeA CRF neurons will result in similar functional connectivity patterns as observed following multiple CIE exposures.